DESCRIPTION: (Applicant's abstract) Determining how cells sense and adapt to fluctuating O2 levels in their environment is a fundamental problem in biology. For many prokaryotic and eucaryotic organism, the sensing of oxygen levels is essential to ensure an adequate supply of energy as well as to avoid the toxic effects of oxygen. In recent years, it has been shown that many members of the FNR family of transcription factors that play a key role in the anaerobic lifestyle of a wide group of prokaryotes, function in oxygen sensing. Thus by deciphering how FNAR activity responds to oxygen availability, we will obtain fundamental information on a process that is critical to the growth and survival of facultative microbes. In addition, our studies should provide key insights into some general properties of oxygen sensing that can be applied to both eucaryotic and prokaryotic cells. In the best-studies case of Escherichia coli, FNR contains a [4Fe-4S]+2 cluster that is required for dimerization and site-specific DNA binding. This [4Fe-4S]+2 cluster is oxygen sensitive and its conversion to a [2Fe-21S]+2 decreases dimerization and DNA binding in vitro. To explain how FNR activity is regulated in vivo, we propose that FNR is largely active under anaerobic conditions because the [4Fe-4S]+2 cluster is stable whereas, in the presence of oxygen, we propose that FNR is largely inactive due to its conversion to a [2Fe-2S]+2 intermediate or possibly an apo-FNR form that lacks a cluster. To determine whether the [4Fe-4S]+2 cluster conversion is sufficient to explain how FNR is inactivated under aerobic conditions in vivo, we will define the pathway of FNR inactivation in vivo. To determine how the presence of the [4Fe-4S]+2 cluster increases FNR activity, we will test the hypothesis that the [4Fe-4S]+2 cluster is required to achieve a conformation that is competent for dimerization. To further define the role of FNR as a global regulator of transcription we will characterize the regions of FNR involved in transcription activation. Our studies should rovide insights into conserved regulatory strategies for sensing changes in oxygen tension by a wide variety of prokaryotes including several pathogenic organisms.